A flow cytometer for use in medical and biological fields includes a fluorescence detection device that receives fluorescence emitted from a fluorochrome in a measurement object irradiated with laser light and identifies the type of measurement object. In the field of medicine, research on the interaction between proteins such as biological binding is being actively conducted. Particularly, research on the interaction between proteins using measurement of fluorescence resonance energy transfer (FRET) is being vigorously pursued. FRET is conventionally measured based on a change in fluorescence intensity, but in recent years, various flow cytometers that analyze measurement objects by utilizing difference in fluorescence relaxation time (fluorescence lifetime) have been proposed.
In a general flow cytometer, a flow is formed by allowing a suspension liquid containing measurement objects, such as biological substances (e.g., proteins, cells, DNA, RNA, or enzymes), labeled with fluorochromes (fluorescent reagents) to flow in a sheath liquid flowing under pressure through a tube at a flow rate of 10 m/sec or less. The flow cytometer receives fluorescence emitted from a fluorochrome attached to each of the measurement objects irradiated with laser light in the flow, and identifies the measurement object by identifying the fluorescence as a label.
Such a flow cytometer can measure, for example, the relative amount of a measurement object, such as DNA, RNA, an enzyme, or a protein, contained in an individual cell and analyze the property of the measurement object in a short period of time. Further, a cell sorter or the like is used to sort only identified cells or chromosomes in the living state in a short period of time after identifying a specific type of cell or chromosome based on fluorescence.
In this case, it is necessary to accurately identify a larger number of measurement objects in a short period of time based on the information of fluorescence.
Patent Document 1 discloses a device and method for identifying individual particles or cells labeled with different fluorochromes based on the lifetime (fluorescence relaxation time) of their fluorescence.
According to the Patent Document 1, laser light whose intensity has been modulated by a modulation signal from a modulator is emitted from a light source toward an irradiation port of a flow chamber to illuminate an individual particle or cell. Fluorescence emitted from an individual particle or cell is converted into a fluorescent signal by a photodetector and sent to two mixers.
On the other hand, the modulation signal from the modulator is sent to the two mixers via a variable phase shifter. At this time, the phase of the modulation signal to be supplied to one of the mixers is shifted by 90 degrees by a 90 degrees-phase shifter that shifts the phase of a signal by 90 degrees, and the phase of the modulation signal to be supplied to the other mixer is not shifted and therefore the modulation signal from the modulator is directly sent to the other mixer.
The fluorescent signal and the modulation signal sent to each of the mixers in such a manner as described above are mixed and passed through a low-pass filter to obtain a real part component and an imaginary part component which are information about the phase of the fluorescent signal. From the ratio between the real part component and the imaginary part component, fluorescence lifetime is calculated.
The Patent Document 1 describes that the flow cytometer can identify individual particles or cells based on the lifetime of their fluorescence.
In addition to the Patent Document 1, Patent Documents 2 and 3 also each disclose a flow cytometer that identifies individual particles or cells by determining the lifetime of their fluorescence from the phase delay of the fluorescence.